There are over twelve million smokeless tobacco (snuff or chewing tobacco) users in the U.S. today, many of them young. Epidemiological studies have indicated that smokeless tobacco usage greatly increases the risk for cancer of the oral cavity. Smokeless tobacco users constitute a nearly unique group in that they represent a large population exposed to high levels of a mixture that contains only one or two major known carcinogens. The important carcinogens thus far identified are 4- (methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN), (both are also components of cigarette smoke), with current evidence suggesting that the former is more likely to be carcinogenic in the oral cavity. Both compounds require oxidative metabolism at the carbon atoms alpha to the N-nitroso group to exert their carcinogenic effects. If these compounds can be established as carcinogens in humans, a number practical applications might follow (e.g., altering the curing process to reduce levels of NNK and NNN, formulating smokeless tobacco products to contain specific inhibitors of NNK and NNN metabolism, requiring snuff manufacturers to indicate levels of NNK and NNN, altering certain lifestyle factors ((e.g. diet)) that might affect metabolism of NNK and NNN, etc.). In addition, intake of NNN and NNK among smokeless tobacco users could be determined, and the dose-response curve for carcinogenesis in a large population of humans could be established - a result with considerable application for toxicological extrapolations. Although NNK and NNN are established carcinogens in experimental animals, and can be metabolized by rat oral tissue, a critical link in establishing them as carcinogens in humans is lacking- they have not been shown to be metabolized by human oral tissues. This project will address that problem. The studies which are planned will measure: 1) biotransformation of NNK by human oral tissue (gingival and buccal tissue homogenates) using a radiometric assay which monitors formaldehyde and methanol (two alpha-oxidative metabolites, 2) K ad V for selected oral tissue samples, 3) metabolism of NNK and NNN to mutagens in Salmonella by human oral tissue. In addition, a key activity (06-methylguanine-DNA methyltransferase) involved in the repair of DNA damage induced by NNK, will be measured in human oral tissue. Finally, lifestyle factors (e.g. smoking, alcohol or smokeless tobacco usage) will be correlated with rates of metabolism of NNK or NNN, and with MT by human oral tissue, to try and identify risk factors or protective factors affecting genotoxic effects of NNK and NNN.